This application claims priority to application no. PCT/JP98/03988, filed Sep. 4, 1998, which claims the priority of Japanese application no. 9-240672, filed Sep. 5, 1997, which are both incorporated herein by reference.
The present invention relates to a fluorescence polarization method for analyzing an assay-object in a sample. In particular, the present invention relates to a fluorescence polarization method useful in analyzing a bacteria or a virus in a sample. The present invention is useful in the fields of art of medical diagnosis, environmental assay, and food control relevant to food poisoning and infectious diseases.
The fluorescence polarization method is known in the art as a method for assaying a substance in a sample. The method is based on the principle that when a fluorescent-labeled compound is excited by linearly polarized light, the fluorescence emitted from the compound has a degree of polarization which is in proportion to the molecular weight thereof.
As a fluorescence polarization method which has been developed, there is a fluorescence polarization immunossay based on an antigen-antibody reaction.
For example, U.S. Pat. No. 4,902,630 discloses an assay method (FIG. 8 is a concept diagram showing the assay principle) in which a body fluid (particularly, a blood) containing CRP (5), an assay-object, is added to a mixed solution which contains: a xe2x80x9ctracerxe2x80x9d (6) obtained by binding fluorescein, a fluorochrome, to C-reactive protein (CRP); and an antibody (4) which specifically binds to CRP. CRP (5) in the sample is assayed based on competition between the tracer (6) and CRP (5) for the antibody (4) in the mixed solution. Since this assay system is based on a competitive reaction, it requires a two-step binding reaction, thereby complicating the assay operation. Moreover, fluorescein used in the assay has a short lifetime of the fluorescence, and thus it is difficult to apply it in an assay for a high-molecular-weight substance.
Japanese Patent Application No. 62-38363 discloses an immunoassay apparatus for an assay of an antigen or antibody utilizing an immunoreaction. It is taught that a fluorescent-labeled antibody can be used in the assay. However, the application describes no specific embodiment of the assay. As an assay-object, it only accounts for a substance, such as a therapeutic drug existing in the blood (e.g., digoxin), whose molecular weight is clearly lower than that of a coexisting protein.
Thus, there has been a demand for development of a fluorescence polarization method with a simple assay reaction system and an easy assay operation, and particularly a method which is suitable for an assay of a high-molecular-weight substance.
An object of the present invention is to provide a fluorescence polarization method for analyzing an assay-object contained in a sample by measuring a change in the degree of the fluorescence polarization thereof, particularly a method which is suitable for an assaying of a high-molecular-weight substance. Another object of the present invention is to provide a reagent for analyzing an assay-object contained in a sample utilizing a fluorescence polarization method.
The present invention relates to a fluorescence polarization method for analyzing an assay-object in a sample, the method including the steps of: (a) providing a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome(s), wherein the protein is an antibody, a receptor or an inhibitor, which is capable of specifically binding to the assay-object; (b) allowing the fluorescent-labeled protein to bind to the assay-object; and (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled protein by its binding to the assay-object.
In the above-described method, the antibody capable of specifically binding to the assay-object may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a Fab antibody or a (Fab)2 antibody.
In the above-described method, the assay-object may be a biological substance; a microorganism including a bacteria; a virus; a drug; an environmental pollutant or an abused drug. The biological substance may be a peptide, a protein, a lipid, a saccharide or a nucleic acid. The protein as a biological substance may have a molecular weight of 500,000 or more.
The protein as a biological substance may be an antibody, a hormone, an inflammation marker, a coagulation factor, an apolipoprotein, a high density lipoprotein (HDL), a low density lipoprotein (LDL), a glycosylated albumin, a glycosylated hemoglobin, a hemoglobin, a cancer marker or an enzyme.
The hormone may be chorionic gonadotropin, thyroid-stimulating hormone, progesterone, follicular forming hormone, parathyroid-stimulating hormone, adrenocorticotropic hormone, or insulin.
The inflammation marker may be C-reactive protein (CRP), xcex11-antitrypsin (xcex11-AT), xcex11-antichymotrypsin (xcex11-X), xcex11-acid glycoprotein (xcex11-AG), haptoglobin (Hp), ceruloplasmin (Cp), the 9th component of complement (C9), the 4th component of complement (C4), the 3rd component of complement (C3), complement factor B (B), fibrinogen (Fbg), serum amyloid A (SAA), C1 inhibitor (C1I), a sialoglycoprotein (i.e., a glycoprotein to which a sialic acid is bound), an acid-soluble protein (ASP) or an immunosuppressive acidic protein (IAP).
The present invention also relates to a reagent for use in a fluorescence polarization method for analyzing an assay-object in a sample, the reagent including a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome, wherein the protein is an antibody, a receptor or an inhibitor, which is capable of specifically binding to the assay-object.
The present invention further relates to a fluorescence polarization method for analyzing a bacteria or a virus in a sample, the method including the steps of: (a) providing a fluorescent-labeled antibody in which an antibody is covalently bound to a fluorochrome, wherein the antibody is capable of specifically binding to the bacteria or the virus; (b) allowing the fluorescent-labeled antibody to bind to the bacteria or the virus; and (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled antibody by its binding to the bacteria or the virus.
In the above-described method, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a Fab antibody or a (Fab)2 antibody.
In the above-described method, the bacteria may be selected from the group consisting of Rhodospirillaceae, Chromatiaceae, Chlorobiaceae, Myxococcaceae, Archangiaceae, Cystobacteraceae, Polyangiaceae, Cytophagaceae, Beggiatoaceae, Simonsiellaceae, Leucotrichaceae, Achromatiaceae, Pelonemataceae, Spirochaetaceae, Spirillaceae, Pseudomonadaceae, Azotobacteraceae, Rhizobiaceae, Methylomonadaceae, Halobacteriaceae, Enterobacteriaceae, Vibrionaceae, Bacteroidaceae, Neisseriaceae, Veillonellaceae, Organisms oxidizing ammonia or nitrite, Organisms metabolizing sulfer and sulfer compounds, Organisms depositing iron and/or manganese oxides, Siderocapsaceae, Methanobacteriaceae, Aerobic and/or facultatively anaerobic Micrococcaceae, Streptococcaceae, Anaerobic Peptococcaceae, Bacillaceae, Lactobacillaceae, Coryneform group of bacteria, Propionibacteriaceae, Actinomycetaceae, Mycobacteriaceae, Frankiaceae, Actinoplanaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae, Rickettsiaceae, Bartonellaceae, Anaplasmataceae, Chlamydiaceae, Mycoplasmataceae and Acholeplasmataceae.
In the above-described method, the virus may be selected from the group consisting of Enterovirus, Cardiovirus, Rhinovirus, Aphthovirus, Calicivirus, Orbivirus, Reovirus, Rotavirus, Abibirnavirus, Piscibirnavirus, Entomobirnavirus, Alphavirus, Rubivirus, Pestivirus, Flavivirus, Influenzavirus, Pneumovirus, Paramyxovirus, Morbillivirus, Vesiculovirus, Lyssavirus, Coronavirus, Bunyavirus, Arenavirus, Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus and Hepatitis C virus.
In the fluorescence polarization method of the present invention, fluorochrome may have a functional group which can bind to a primary, secondary or tertiary amino group, a carboxyl group, a thiol group, a phenyl group, a phenol group or a hydroxyl group. The lifetime of the fluorescence of the fluorochrome may be in the range of 10 nanoseconds to 200 nanoseconds. The fluorochrome may have a skeletal structure of rhodamine, pyrene, dialkylaminonaphthalene or cyanin.
The present invention also relates to a reagent for use in a fluorescence polarization method for analyzing a bacteria or a virus in a sample, the reagent including a fluorescent-labeled antibody in which an antibody is covalently bound to a fluorochrome, wherein the antibody is capable of specifically binding to the assay-object.